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SAM 908.07

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 908

Supplemental Assay Method for Sterility Testing of Live Viral Vaccines and 

Master Seed Virus Samples

Date: May 12, 2023

Number: SAM 908.07

Supersedes: SAM 908.06, July 28, 2018

Standard Requirement: 9 CFR, part 113.27(a) and (c)

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product 

by USDA and does not imply its approval to the exclusion of other products that may be suitable.
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the samples

4. Performance of the Test

5. Examination of the Test Vessels

6. Interpretation of the Test Results

7. Record and Report of Test Results

8. References

9. Summary of Revisions

Appendices
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1. Introduction

This Supplemental Assay Method (SAM) describes the test procedure used to detect viable 

bacteria and fungi in all live viral vaccines and Master Seed Virus samples, per title 9, Code of

Federal Regulations (9 CFR), parts 113.27(a) and (c). In the presence of contaminating 

extraneous agents, the medium will be rendered turbid by macroscopic examination.

2. Materials 

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 30°- 35°C incubator

2.1.2 20°- 25°C incubator

2.1.3 Laminar-flow Class II biosafety cabinet (BSC)

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Bacillus subtilis (American Type Culture Collection (ATCC) #6633) or 

equivalent organism as specified in the current United States Pharmacopoeia 

(USP)

2.2.2 Issatchenkia orientalis (ATCC #6258) or equivalent organism as 

specified in the current USP

2.2.3 Soybean Casein Digest Medium (SCDM) or Trypticase Soy Broth (TSB)

(National Centers for Animal Health (NCAH) Media #10423) (Appendix I)

2.2.4 Trypticase Soy Agar (TSA) plates, NCAH Media #10487 (Appendix II)

2.2.5 Glassware: tubes and flasks containing test media 

2.2.6 Sterile water in serum vials

2.2.7 Lab coat or sterile sleeves and gloves

2.2.8 70% ethanol
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2.2.9 Sterile gauze pads, 4 x 4-inch

2.2.10 Sterile syringes with needles

2.2.11 Vacutainer® needles 

2.2.12 Sterile pipettes, individually packaged

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques and preparation, proper handling, and disposal of 

biological agents, reagents, and chemicals. Personnel must also have knowledge of safe 

operating procedures and policies as well as training in the operation of the necessary 

laboratory equipment listed in Section 2.1.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Turn on the BSC at the beginning of the work week and leave on all week.

3.2.3 Monitor the temperature of incubators, freezers, and coolers according to 

SOPs.

3.3 Preparation of reagents/control procedures

3.3.1 Bacillus subtilis stock culture is prepared according to the manufacturer’s 

instructions and titrated to determine colony forming unit (CFU) concentration.

3.3.2 Issatchenkia orientalis stock culture is prepared according to the 

manufacturer’s instructions and titrated to CFU concentration.

3.3.3 Dilution of Preservative Screening (Eleventh Vessel Positive Control): 

For each serial tested, inoculate an additional container of media for each 

incubation temperture with 0.2 mL of sample and approximately 100 CFU of the 

appropriate indicator organism (Sections 2.2.1 and 2.2.2). This control is 

conducted to confirm the ratio of inoculum to medium that will result in sufficient 

dilution of the product to prevent bacteriostatic and fungistatic activity according 

to 9 CFR 113.25(d).
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3.3.4 Maximum Medium Volume Limits per Vessel: The maximum volume of 

media per vessel used will not exceed 500 mL as these large volumes are 

hazardous when removing from the autoclave. Volumes greater than 500 mL will 

be divided evenly into two vessels and the volume of inoculum will be divided 

accordingly.

3.3.5 Negative Control: Include 20 uninoculated test vessels of media to 

confirm the sterility of the media batch according to 9 CFR 113.25(c). Incubate 

10 representative test vessels at each incubation temperature for 14 days.

3.4 Preparation of the samples

3.4.1 Follow Section V.A. of the Outline of Production (OP) for final product(s) 

to determine the volume of media that should be aliquoted into each vessel. 

Master Seed Virus (MSV) samples are tested with a minimum of 120 mL of TSB 

at 30°- 35°C and 40 mL of TSB at 20°- 25°C. Order a sufficient volume of media 

to accommodate the test vessels, positive controls, negative controls, and extra 

vessels for potential subcultures.

3.4.2 For products without accompanying diluent, order sterile purified water in 

serum vials in volumes specified on the product label or in the OP.

3.4.3 Ten vials of final product and a minimum of 4.5 mL of MSV are required 

for sterility testing.

4. Performance of the Test

4.1 Dress in a clean lab coat or sterile sleeves and gloves to perform sterility testing.

4.2 Wipe down the interior surfaces of the BSC used for testing with 70% ethanol

immediately prior to use and between testing each serial or lot.

4.3 Place the necessary testing materials (syringes, Vacutainer® needles, 4 x 4-inch

gauze squares, etc.), test media, and the product to be tested in the BSC.

4.4 Swab the top of each container of product, diluent, and water with gauze soaked 

in 70% ethanol. 

4.5 For live viral vaccines, rehydrate 10 vials of each lyophilized serial with water or 

accompanying diluent using a syringe and needle or Vacutainer® needle. Rehydrate 

products for mass inoculation with sterile water at a rate of 30 mL per 1000 doses or as

specified on the product label or OP. If necessary, MSV is rehydrated with TSB (SCDM).
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4.6 Withdraw product from each sample container with a new sterile syringe and 

needle. Dispense a 0.2 mL aliquot from each vial of the rehydrated liquid or thawed 

frozen liquid product into two test vessels (one tube per incubation temperature). 

Dispense 0.2 mL of rehydrated or thawed MSV samples among the 20 test vessels. Swirl 

each test vessel after inoculating with sample to distribute the product in the media. 

4.7 Inoculate two additional vessels of media with 0.2 mL of product to serve as the 

11th vessel positive controls (see Section 3.3.3). The product sample for these control 

vessels may be obtained from any of the previously used containers or from an 11th vial 

of product. Set the 11th vessels to the side and continue with the testing.

Note: The 11th vessel positive control is not conducted with MSV.

4.8 When all of the test vessels have been inoculated with product, prepare the 

negative control for the testing session (see Section 3.3.5). 

4.9 Once the sterility portion of the test has been completed, prepare the positive

control organisms in an area that is separate and apart from the clean area where the 

sterility test was conducted (see Sections 3.3.1 and 3.3.2).

4.9.1 Inoculate approximately 100 CFU of the appropriate indicator organism

into the vessels prepared in Section 4.7 and swirl the vessel to distribute the 

organism in the medium. 

4.9.2 After the 11th test vessels have been inoculated with the indicator 

organisms, inoculate one TSA plate per indicator organism with a representative

volume of inoculum. This plate count serves to demonstrate that the appropriate 

number of viable organisms were added to the test vessel.

4.10 Incubate the test vessels and the representative controls at the appropriate

incubating temperature. Incubate all vessels for 14 days. Incubate the TSA plates 

containing the indicator organisms for a maximum of 7 days.

4.11 Wipe down the interior of the BSC and counter tops with 70% ethanol at the end 

of the testing session. Discard biological samples and contaminated materials according 

to SOPs.
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5. Examination of the Test Vessels

5.1 Examine all test vessel medium for cloudiness or turbidity at least once during 

days 7 to 11 of the incubation period and on day 14. 

5.1.1 Determine if the cause of cloudiness or turbidity is due to microbial 

growth by conducting a Gram stain on an aliquot of the culture.

5.1.2 If growth in a vessel cannot be reliably determined by Gram stain, 

subculture the contents of the test vessel by aseptically transferring 1.0 mL from 

the test vessel in question to 40.0 mL of fresh test media. Swirl the vessel to mix 

the inoculum into the media and incubate for a minimum of 3 days. In addition, 

subculture the test vessel culture onto two plates of blood agar and TSA and 

streak for isolation. Incubate one plate of each at both incubation temperatures for 

3 days.

5.2 On day 14, vessels without growth are considered negative. 

5.3 By day 14, any vessels with bacterial or fungal growth confirmed by Gram stain 

and/or growth in the subcultures are considered positive for extraneous growth. The 

contaminant will also be identified by Matrix-Assisted Laser Desorption/Ionization 

Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

6. Interpretation of the Test Results

6.1 Criteria for a valid test:

6.1.1 There must be no growth in any of the Negative Control vessels.

6.1.2 For final products tested for dilution of preservative at the Center for 

Veterinary Biologics, the TSA plates containing B. subtilis must contain an 

average count of 93-128 CFU and the TSA plates containing I. orientalis must 

contain an average count of 76-124 CFU. 

Note: A range of approximately 100 CFU should be determined at each biologics 

manufacturer facility for each new positive control lot. 

6.1.3 For final products tested for the dilution of preservative, growth of the 

indicator organism must be observed in the 11th vessel positive control.

6.1.4 If these criteria are not met, the test is considered invalid or a no test (NT). 

Products may be reported and released with a NT result if there is no reason to 

suspect an unsatisfactory sterility result for that product.
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6.2 For final product: 

 6.2.1 If extraneous growth is found in 2 or 3 of the 20 test vessels of the initial 

test, one retest (RT) may be conducted using 20 unopened final container 

samples. 

 6.2.2 If no extraneous growth is detected in 19 or 20 test vessels of the initial 

test or, 39 or 40 test vessels of the RT, the serial is satisfactory (SAT). 

 6.2.3 If extraneous growth is found in 4 or more of the 20 test vessels of the 

initial test or, 2 or more of the 40 test vessels of the RT, the serial is unsatisfactory 

(UNSAT). 

 6.2.4 If there is no growth in the 11th vessel positive control(s), there will be a 3-

week delay before the test results are reported (Center for Veterinary Biologics 

Notice 09-02, 

http://www.aphis.usda.gov/animal_health/vet_biologics/publications/notice_09_0

2.pdf) while a dilution of preservative study will be conducted on the serial

according to 9 CFR 113.25(d). If the volume of media listed in the OP for sterility 

testing of the serial indicates interference in the 9 CFR 113.25(d) test, this testing 

will be reported as UNSAT and the sterility test result will be reported as a NT. 

6.3 For MSV: 

 6.3.1 If extraneous growth is observed in any test vessel of the initial test, one 

RT may be conducted using a new sample of MSV. 

 6.3.2 If extraneous growth is found in any test vessel of the final test, the lot of 

MSV is UNSAT.

7. Report of Test Results

Record and report the test results as described by SOPs. 

8. References

8.1 Title 9, Code of Federal Regulations, parts 113.25 and 113.27, U.S. Government 

Printing Office, Washington, DC. 

8.2 The U.S. Pharmacopoeia, 1985, Vol. 21, pp. 1151-1160, Mack Publishing Co., 

Easton, PA. 
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8.3 Kurtzman, C. P., C. J. Robnett, and E. Basehoar-Powers. 2008. Phylogenetic 

relationships among species of Pichia, Issatchenkia and Williopsis determined from 

multigene sequence analysis, and the proposal of Barnettozyma genera novel, Lindnera 

genera novel and Wickerhamomyces genera novel. FEMS Yeast Res 8:939-54. 

9. Summary of Revisions 

Version .07 

• Updated cover page.

• Adjusted Bacillus subtilis acceptable validity range. 

Version .06 

• Updated cover page.

• Updated Sections 2 through 6.

Version .05 

• The Bacteriology Section Leader has been updated. 

• Updated Sections 2.2, 3.3, and 4. 

• Deleted Appendix III (Blood Agar base with 5% bovine blood – NVSL Media 

#10006).

Version .04 

• Clarified interpretation of test results in Section 6.2. 

Version .03

• Revised to include dilution of preservative requirements cited in CVB Notice 09-02. 

• The Contact phone number has been updated. 

• Sections 3, 4, 5, and 6: These sections have been updated to reflect current practices.

• 8.3: Reference added for name change of Candida krusei to Issatchenkia orientalis. 

• Appendices: Updated media storage limits to be in compliance with 9 CFR 

113.25(b). 

Version .02 

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail and the following changes were made to the document: 

• The Contact has been changed from Dolores Strum to Sophia Campbell. 

• 2.1 The Bunsen burner has been removed from the list of equipment that is needed 

for the test.

• 3.3.2: This section has been added to describe the 11th vessel positive control. 

• 3.3.6: This section has been added to clarify media volume limits per vessel used in 

testing. Also added a note on the safety hazard when removing volumes > 500 mL 

from the autoclave. 
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• 3.4.3 Determination of the volume of test media needed for each serial to be tested 

on the dilution of preservative computer file using Lotus Approach 97 has been 

removed. 

• 4.1 Disinfection of the vials of the serials of biologic to be tested with 0.05% Germ 

Warfare using a Clean-Pal or equivalent has been removed. 

• 4.8 Instead of “dehydrating” the “desiccated” vials, the instruction has changed to 

“rehydrating” the “lyophilized” vials. 

• 5.1: The section has been rewritten to indicate that tubes chosen for subculturing are 

at the technician’s discretion rather than randomly. 

• 5.4: Clarification on how to interpret the 11th vessel positive control results has been 

added, along with information on follow-up testing. 

• 5.6: Additional information regarding contaminant labeling has been added. 

• Media storage information has been added to the Appendix.

Version .01 

The information contained in this document was previously available as a protocol 

(STPRO0270.01 dated September 9, 1996). The document was rewritten to meet the current 

NVSL/CVB QA requirements, to clarify practices currently in use in the CVB-L, and to provide 

additional detail. No significant changes were made from the previous protocol.
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Appendix I

Soybean Casein Digest Medium (SCDM) or Trypticase Soy Broth (TSB), NCAH Media 

#10423

Trypticase Soy Broth 30 g

QH2O 1000 mL

Autoclave 20 minutes at ≥121°C. Store at 20°- 25°C for up to 90 days.

Note: TSB and SCDM are synonymous mediums.

Appendix II

Trypticase Soy Agar (TSA), NCAH Media #10487

Trypticase Soy Agar 40 g

QH2O 1000 mL

Boil to dissolve and autoclave for 15 minutes at ≥121°C. Store at 2°- 7°C for up 

to 90 days.